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RNA degradation is fundamental for cellular homeostasis. The process is carried out by various classes of endolytic and exolytic enzymes that together degrade an RNA polymer to mono-ribonucleotides. Within the exoribonucleases, nano-RNases play a unique role as they act on the smallest breakdown products and hence catalyze the final steps in the process. We recently showed that oligoribonuclease (Orn) acts as a dedicated diribonuclease, defining the ultimate step in RNA degradation that is crucial for cellular fitness (Kim et al., 2019). Whether such a specific activity exists in organisms that lack Orn-type exoribonucleases remained unclear. Through quantitative structure-function analyses, we show here that NrnC-type RNases share this narrow substrate length preference with Orn. Although NrnC and Orn employ similar structural features that distinguish these two classes of dinucleases from other exonucleases, the key determinants for dinuclease activity are realized through distinct structural scaffolds. The structures, together with comparative genomic analyses of the phylogeny of DEDD-type exoribonucleases, indicate convergent evolution as the mechanism of how dinuclease activity emerged repeatedly in various organisms. The evolutionary pressure to maintain dinuclease activity further underlines the important role these analogous proteins play for cell growth. 

ABSTRACT The dinucleotide second messenger c-di-GMP has emerged as a central regulator of reversible cell attachment during bacterial biofilm formation. A prominent cell adhesion mechanism first identified in pseudomonads combines two c-di-GMP-mediated processes: transcription of a large adhesin and its cell surface display via posttranslational proteolytic control. Here, we characterize an orthologous c-di-GMP effector system and show that it is operational in Vibrio cholerae , where it regulates two distinct classes of adhesins. Through structural analyses, we reveal a conserved autoinhibition mechanism of the c-di-GMP receptor that controls adhesin proteolysis and present a structure of a c-di-GMP-bound receptor module. We further establish functionality of the periplasmic protease controlled by the receptor against the two adhesins. Finally, transcription and functional assays identify physiological roles of both c-di-GMP-regulated adhesins in surface attachment and biofilm formation. Together, our studies highlight the conservation of a highly efficient signaling effector circuit for the control of cell surface adhesin expression and its versatility by revealing strain-specific variations. IMPORTANCE Vibrio cholerae , the causative agent of the diarrheal disease cholera, benefits from a sessile biofilm lifestyle that enhances survival outside the host but also contributes to host colonization and infectivity. The bacterial second messenger c-di-GMP has been identified as a central regulator of biofilm formation, including in V. cholerae ; however, our understanding of the pathways that contribute to this process is incomplete. Here, we define a conserved signaling system that controls the stability of large adhesion proteins at the cell surface of V. cholerae , which are important for cell attachment and biofilm formation. Insight into the regulatory circuit underlying biofilm formation may inform targeted strategies to interfere with a process that renders this bacterium remarkably adaptable to changing environments.